The importance of long-chain polyunsaturated fatty acids of the ω-3 type, the acids (all cis)-5, 8, 11, 14, 17 eicosapentaenoic, hereinafter EPA, and (all cis)-4, 7, 10, 13, 16, 19 docosahexaenoic, hereinafter DHA, for ingredients of food or pharmaceutical products is well known and documented because of their usefulness, among others, to prevent arteriosclerosis and cardiovascular diseases, alleviate inflammatory conditions and delay the growth of tumors. As a consequence, experts recommend a daily intake of said fatty acids ranging between 0.5 and 10 g.
One of the richest sources of EPA and DHA are fish oils of different origin such as sardines, jack mackerel, anchovy, salmon, codfish and others. Typically, the combined content of EPA and DHA in said oils is around 10 to 35% in weight. Consequently, the first attempts to provide food supplements and pharmaceuticals rich in EPA and DHA were based on refined fish oils in order to remove their characteristic unpleasant odor and flavor, for their utilization as ingredients for food or pharmaceutical product suitable for human consumption. These refining processes resorted mainly to the classic processes for vegetable oil refining oils and specific adaptations of said processes to the raw material concerned (Lindsay, U.S. Pat. No. 4,915,876; Chang, U.S. Pat. No. 4,874,629; Marschner, U.S. Pat. No. 4,804,555; Stage, U.S. Pat. No. 4,599,143; Merck, U.S. Pat. No. 4,838,997).
Nevertheless, current attempts to provide EPA and DHA from refined marine oils suitable as ingredient for food and pharmaceutical product, have not been successful to provide a product whose organoleptic properties were acceptable and were free of the typical secondary effects such as gastric reflux, stomach and skin irritation and meteorism, among others. These effects are accentuated when EPA and DHA are consumed in quantities over 1 g, that is, doses equivalent to about 5 g of fish oil, producing the mentioned secondary effects in the consumer.
Consequently, the efforts to provide EPA and DHA have been directed towards the production of concentrates of these acids from marine oils. These concentrates may contain between 40 to 95% of EPA and DHA in weight, either in the form of free acids, in the form of esters, typically ethyl esters or mono, di or triglycerides. The aim of these processes is to provide concentrates of EPA and DHA that have better organoleptic properties of flavor, odor and color, that can be used directly in products for therapeutic use in humans, as an active pharmaceutical ingredient or as food ingredients in general. Nevertheless, the state of art does not provide processes capable of providing products fulfilling the characterisitics of having good sensory properties, long term storage and oxidative stability to keep their desirable organoleptic properties over time, that is, products in which reversion in time to fishy odor and flavor do not occur and which lack of the typical secondary effects of the marine oils and their derivatives, such as gastric reflux, flattulence, allergy among others.
Concentrates of EPA and DHA currently available commercially are not used directly as food ingredient, but instead they are utilized in form of syrups in which the flavor has been camouflaged or in form of sugar-coated or micro-encapsulated pills, all this with the purpose of hiding or minimizing the undesirable flavor and odor that develop in time in said products. Additionally, these concentrates are not suitable either for therapeutic uses that normally require relatively high doses of EPA or DHA, several grams per day, because at these doses the undesirable secondary effects of the concentrates are even more accentuated.
Another approach for providing EPA or DHA for human consumption derived from marine oils has been the development of processes to obtain pure EPA or DHA, as is disclosed in U.S. Pat. No. 6,846,942. Nevertheless, obtaining pure EPA or DHA means passing first through a step where a mixture of EPA and DHA is obtained; commercially there does not seem to be an advantage to this approach, and as can be observed in the documents of Table 1, most of the processes disclosed deal with the preparation of concentrates containing EPA and DHA either in the form of free acids or in the form of esters.
Numerous processes disclosed in the prior art, addressed to obtain of concentrates of ω-3 fatty acids from oil are shown in Table 1.
European patent No 0 409 903 discloses a process for preparing mixtures containing EPA and DHA from animal or vegetable oils. The process comprises the steps of saponifying the raw material, the animal or vegetable oil, acidifying the saponified mixture immediately and then extracting the acids formed with petroleum ether until exhaustion. The extracts are then washed with water, the solvent removed and the residue subjected to one or more steps of molecular distillation at a pressure of 0.133 Pa and a temperature between 110-120° C. A distillate is obtained that contains between 35 and 90% of EPA and DHA.
TABLE 1Patents and Patent Applications for methods orprocesses for the production of DHA and EPADocumentTitle20030027865Method for isolating highly purified fatty acids usingcrystallization.20040022923Marine oils with reduced levels of contaminants20040236128Method for preparing pure EPA and pure DHA20050201997Promoter of elimination of dioxins20050256326Process for decreasing environmental contaminants inan oil or fat, a fluid for decreasing volatile environ-mental contaminants, a food supplement20080268117Method for purifing oils that contain EPA and DHA3,682,993Purification of oils4,554,107Refined fish oils and the process for producing them4,599,143Process for physical deodorizing and/or refining ofoils, fats, edible organic esters with a high boilingpoint.4,623,488Refined fish oils and the process for producing them4,675,132Polyunsaturated fatty acids of fish oils4,692,280Purification of fish oils4,792,418Method for extraction and purification of polyun-saturated fatty acids originating from natural sources.4,838,997Process for deodorizing triglyceride oils4,855,154Process for deodorizing marine oils4,874,629Purification of fish oil4,915,876Process for deodorizing and stabilizing polyun-saturated oils4,966,734Deodorizing of mixtures of fatty esters5,006,281Process for the production of animal oils5,023,100Fish oil5,130,061Process for the extraction of esters from polyun-saturated fatty acids from fish oils5,679,809Concentrate of ethyl esters of polyunsaturatedfatty acids5,693,835Fish oil with less aroma of fish and a method for itspreparation5,945,318Refining of compositions of oil6,190,715Process for producing refined edible fish oil from theherring and other similar fish that contain long-chainomega-3 fatty acids.6,204,401Purification of glycerides of polyunsaturated fatty acids6,214,396Method and plant for extraction of fish oil andresulting products6,261,608Method for the preparation of refined fish oil6,528,669Recuperation of polyunsaturated fatty acids startingwith urea adducts6,537,787Enzymatic methods for enriching with polyunsaturatedfatty acids6,664,405Method for isolating highly purified unsaturated fattyacid using crystallization6,846,942Method for preparing pure EPA and pure DHAEP0409903B1Process for preparing polyunsaturated fatty acidsEP0749468B1Refining of oil compositionsEP0968264B1Purification of glycerides of polyunsaturated fatty acidsEP1153114B1Esterification of marine oil catalyzed by lipaseEP1178103A1Purification of raw oil with polyunsaturated fatty acidsEP1202950B1Recuperation of polyunsaturated fatty acids starting withurea adductsEP1996686A1Omega 3JP2007138181Process for preparing material with a high content oflong-chain polyunsaturated fatty acids
U.S. Pat. No. 5,130,061 discloses a process for the preparation a mixture of highly concentrated ethyl esters of EPA and DHA from fish oil. The process disclosed includes the steps of transesterifying fish oil with ethyl alcohol, followed by the extraction of the transesterified product with hexane and the purifying of the extract by chromatography in silica gel. Then, the purified product is subjected to one or more steps of molecular distillation at a pressure of about 0.001 mmHg and temperature between 65 to 70° C. Optionally, prior to the distillation, the product resulting from the chromatography may be crystallized in acetone at −40° C. and then subjected to distillation.
Many of the processes disclosed can provide products with acceptable organoleptic properties but in all of them the above-described secondary effects are produced and the reversion to fishy smell and flavor occurs over time, unlike in the product obtained by means of the process of the present invention that keep its neutral organoleptic properties under storage conditions at room temperature during a period of at least three months and without causing significant secondary effects in the consumer. By neutral organoleptic characteristics we mean a product having acceptable organoleptic properties in the absence of additives to mask the flavor or odor, while acceptable organoleptic characteristics are understood to be a product evaluated by a trained sensory panel composed of at least 9 members evaluating product properties such as appearance, aroma and flavor with a qualification of each parameter equal or greater than 60% of the maximum value of said parameter, and the rancidity property, with a qualification equal to or greater than 80% of the maximum value of said parameter.
In addition to the requirement of acceptable organoleptic properties and stability, the concentrates of EPA and DHA must also comply with a series of regulatory norms with regard to their content of contaminating organic compounds known as Persistent Organic Pollutants (POP) that are chemical substances which persist in the environment, accumulate in the food chain and imply a risk of causing adverse effects to human health and to the environment. Among these contaminants, that currently include 17 substances recognized during the third conference of the Parties to the Stockholm Covenants of May 2007, are derivatives of dioxins, furans, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, etc, whose concentration in the fish oils has been increasing over time, so that efforts are being made to develop processes capable of removing these contaminants from fish oils. Among the Parties to the Stockholm Covenants there are currently strict norms with regard to the maximum permissible limits of the POP in products for human consumption, among which include fish oil and products derived from fish oils. Processes specifically addressed to the removal of the POP are found, among others, in the processes disclosed in patent applications US 2005/0256326 and US 2004/0022923 and international application WO 02/06430. Another group of regulated pollutants are heavy metals such as arsenic, mercury, cadmium, and lead, among others.
The processes for the production of concentrates of EPA and DHA described in European patent No 0 409 903 and in U.S. Pat. No. 5,130,061 do not refer to the problem of the presence of POP. Therefore in order to compare the efficiency for removing contaminants of the processes disclosed with the efficacy of the process of this invention to the same object, the mentioned process were reproduced with raw materials having a known concentration of POP and compared to the products obtained by the process of the present. Results are shown in Comparative Examples 1 and 2.
In U.S. Pat. No. 6,846,946 there is a mention of the problem of polychlorinated biphenyls (PCBs) but no solution is disclosed concerning their removal.
It has been found that, surprisingly, the process of this invention, unlike the processes of the prior art, is capable of providing a product with acceptable organoleptic properties, without producing a reversion to fishy of odor and flavor during a time of storage at room conditions of at least three months and also unlike the processes of the prior art it is also capable of efficiently reducing or eliminating the POP and heavy metals. Additionally, the disclosed process does not cause undesirable cis-trans isomerization of EPA and DHA isomers of unknown metabolic properties, but on the contrary and quite surprisingly, reduce the trans isomers content when they are found in the raw materials.
Pronova BioPharma (www.pronova.com) discloses a process for the production of concentrates of ethyl esters of EPA and DHA for their use as an active pharmaceutical ingredient. In said process, crude fish oil is first desacidified to obtain refined fish oil and this refined fish oil is subjected to a stripping process directed specifically to the removal of contaminants by means of the disclosed process in the US application 2005/0256326. The refined fish oil is obtained is subsequently transesterified with ethyl alcohol. The transesterified product is subjected to several steps of molecular distillation. The distillate is treated with urea, then bleached and redistilled molecularly, obtaining a final product with up to 90% of long-chain ω-3 fatty acids between EPA and DHA. A disadvantage of the process is the possibility of transisomerization during the stripping step. Additionally the commercial product reverts to fishy odor and flavor and all previously mentioned secondary effects can be observed upon ingestion of the product.
The process developed by Napro Pharma (www.napro-pharma.no/production) for the production of a concentrate of ethyl esters of EPA and DHA is similar to the process of Pronova BioPharma, but without the stripping step and the urea treatment step, but the product also reverts to fishy odor and flavor and all previously mentioned secondary effects can be observed upon ingestion of the product.
In comparison with the concentrates of EPA and DHA obtained by the process of the state of art, the concentrates obtained by the invented process have surprising and unexpected advantages over the prior art as it will be evident from the detailed description of the invention. These advantages include the characteristic of a organoleptical neutral and stable product, which lacks of secondary effects and levels of Persistent Organic Pollutants complying with international regulatory norms. Furthermore, as mentioned above the process not only prevents the formation of cis-trans isomers, but on the contrary, in a surprising and unexpected manner, it reduces the concentration of trans isomers when these are present in the raw material. As a result of all these combined characteristics, the product obtained by the process of this invention is especially adequate for use in therapies that require high doses of EPA and DHA and as a food ingredient.